Allosteric regulation of catalytic activity of the serine protease factor VIIa (VIIa) is utilized as a mechanism to control the initiation of the coagulation pathways (Ruf, W. and Dickinson, C. D. (1998) Trends Cardiovasc. Med. 8, 350–356). VIIa circulates in the blood plasma as zymogen as well as the cleaved two chain enzyme (Seligsohn, U., Kasper, C. K., Osterud, B., and Rapaport, S. I. (1979) Blood 53, 828–837), but proteolytic function only ensues upon binding to its cell surface receptor and catalytic cofactor tissue factor (TF). TF has two distinct effects that regulate proteolysis by VIIa. First, TF provides affinity for macromolecular substrate by contributing to an extended exosite together with the bound VIIa γ-carboxyglutamic acid-rich (Gla) domain. This exosite is a binding site for the factor X Gla-domain (Ruf, W., Miles, D. J., Rehemtulla, A., and Edgington, T. S. (1992) J. Biol. Chem. 267, 6375–6381; Huang, Q. L., Neuenschwander, P. F., Rezaie, A. R., and Morrissey, J. H. (1996) J. Biol. Chem. 271, 21752–21757; and Ruf, W., Shobe, J., Rao, S. M., Dickinson, C. D., Olson, A., and Edgington, T. S. (1999) Biochemistry 38, 1957–1966). Second, TF enhances catalytic activity by allosteric effects on the VIIa protease domain. In the absence of cofactor, VIIa has only very low catalytic activity towards small peptidyl substrate mimetics and TF stimulates the amidolytic of VIIa up to 100-fold. However, macromolecular substrate factor X scissile bond catalysis is enhanced >1000-fold (Ruf, W., Rehemtulla, A., Morrissey, J. H., and Edgington, T. S. (1991) J. Biol. Chem. 266, 2158–2166), indicating that cofactor-induced conformational changes may influence extended macromolecular substrate recognition regions in addition to the S1–S3 subsite that is probed by the small substrates.
The low catalytic activity of free VIIa results from a zymogen-like conformation of the enzyme. Upon zymogen cleavage, serine protease domains typically undergo a conformational ordering of loop segments, termed the activation domain (Huber, R. and Bode, W. (1978) Accounts Chem. Res. 11, 114–122), resulting in the formation of an activating canonical salt bridge of Asp343 with the newly generated amino-terminal Ile153. In the absence of cofactor, VIIa shows an increased susceptibility of the amino-terminus to chemical modification (Higashi, S., Nishimura, H., Aita, K., and Iwanaga, S. (1994) J. Biol. Chem. 269, 18891–18898), indicating exposure of Ile153 that can result from an alternative conformation or increased flexibility and disorder in the activation pocket of free VIIa. The structural determinants for the propensity of VIIa to stay in a zymogen-like conformation have not been investigated. Available structures of free and TF-bound VIIa (Banner, D. W., D'Arcy, A., Chène, C., Winkler, F. K., Guha, A., Konigsberg, W. H., Nemerson, Y., and Kirchhofer, D. (1996) Nature 380, 41–46; Zhang, E., St. Charles, R., and Tulinsky, A. (1999) J. Mol. Biol. 285, 2089–2104; Pike, A. C. W., Brzozowski, A. M., Roberts, S. M., Olsen, O. H., and Persson, E. (1999) Proc. Natl. Acad. Sci. USA 96, 8925–8930; Kemball-Cook, G., Johnson, D. J. D., Tuddenham, E. G. D., and Harlos, K. (1999) J. Struct. Biol. 127, 213–223; and Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., and Lazarus, R. A. (2000) Nature 404, 465–470) did not provide mechanistic insight, since in each case the active site of VIIa was occupied with inhibitors that are known to stabilize the Ile153-Asp343 saltbridge (Higashi, S., Matsumoto, N., and Iwanaga, S. (1996) J. Biol. Chem. 271, 26569–26574) and restrict conformational flexibility in the VIIa protease domain (Dickinson, C. D., Shobe, J., and Ruf, W. (1998) J. Mol. Biol. 277, 959–971). Mutational studies also failed to elucidate the basis for the labile enzyme conformation, because the taken approaches mainly probed the active enzyme in the TF-VIIa complex (Dickinson, C. D., Kelly, C. R., and Ruf, W. (1996) Proc. Natl. Acad. Sci. USA 93, 14379–14384).
This study investigates the role of residue Met298 in maintaining the zymogen-like conformation of VIIa. This residue is located within the activation pocket, covering Ile153 upon amino-terminal insertion (Banner, D. W., D'Arcy, A., Chène, C., Winkler, F. K., Guha, A., Konigsberg, W. H., Nemerson, Y., and Kirchhofer, D. (1996) Nature 380, 41–46). The conformation of the 298 side chain can influence the catalytic activity of serine protease domains. In the case of tissue plasminogen activator (tPA) (Renatus, M., Engh, R. A., Stubbs, M. T., Huber, R., Fischer, S., Kohnert, U., and Bode, W. (1997) EMBO J. 16, 4797–4805; and Tackiest, K. and Madison, E. L. (1997) J. Biol. Chem. 272, 28–31) and vampire bat plasminogen activator (Renatus, M., Stubbs, M. T., Huber, R., Bringmann, P., Donner, P., Schleuning, W.-D., and Bode, W. (1997) Biochemistry 36, 13483–13493), Lys298 can substitute for Ile153 to form an activating salt bridge with Asp343, resulting in efficient catalysis in the absence of zymogen cleavage. However, Lys at this position is found in a large number of serine proteases without conferring catalytic activity in the zymogen precursors, indicating that multiple interactions within the activation pocket determine the activation state of serine protease domains. Whereas Lys or other hydrophilic side chains are predominant in serine proteases that undergo spontaneous ordering of the activation pocket upon zymogen cleavage, VIIa has a Met residue in the 298 position. We hypothesized that the side chain property of Met298 is one of the determinants that interfere with the acquisition of full catalytic activity of VIIa upon zymogen cleavage. This study demonstrates that replacement of Met298 with Gln, the side chain found in factor IX, had little effect on the activity of TF-bound VIIa. However, free VIIaGln298 had enhanced catalytic function towards macromolecular and small peptidyl substrates. These experiments thus identify the first residue side chain that is one of the determinants for the zymogen-like conformation of the VIIa protease domain.